Synthesis and Antimicrobial Evaluation of 1,4-Naphthoquinone Derivatives as Potential Antibacterial Agents

Article abstract of DOI:10.1002/open.201900077

1,4-Naphthoquinones are an important class of compounds present in a number of natural products. In this study, a new series of 1,4-naphthoquinone derivatives were synthesized. All the synthesized compounds were tested for in vitro antimicrobial activity.

Full text of DOI:10.1002/open.201900077

DOI: 10.1002/open.201900077
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Synthesis and Antimicrobial Evaluation of
1,4-Naphthoquinone Derivatives as Potential Antibacterial
Agents
Palanisamy Ravichandiran,*[a] Maciej Masłyk,[b] Sunirmal Sheet,[c] Monika Janeczko,[b]
Dhanraj Premnath,[d] Ae Rhan Kim,[e] Byung-Hyun Park,[f] Myung-Kwan Han,[g] and
Dong Jin Yoo*[a]
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1,4-Naphthoquinones are an important class of compounds
present in a number of natural products. In this study, a new
series of 1,4-naphthoquinone derivatives were synthesized. All
the synthesized compounds were tested for in vitro antimicro-
bial activity. In this present investigation, two Gram-positive
and five Gram-negative bacterial strains and one pathogenic
yeast strain were used to determine the antibacterial activity.
Naphthoquinones tested for its antibacterial potencies, among
seven of them displayed better antimicrobial activity against
Staphylococcus aureus (S. aureus; 30–70 μg/mL). Some of the
tested compounds showed moderate to low antimicrobial
activity against Pseudomonas aeruginosa (P. aeruginosa) and
Salmonella bongori (S. bongori; 70–150 μg/mL). In addition,
most active compounds against S. aureus were evaluated for
toxicity to human blood cells using a hemolysis assay. For
better understanding, reactive oxygen species (ROS) generation,
time-kill kinetic study, and apoptosis, necrosis responses were
investigated for three representative compounds.
1. Introduction
almost 25,000 deaths in Europe every year.[1,2] The ESKAPE
pathogens such as Enterococcus faecium (E. faecium), Staph-
ylococcus aureus (S. aureus), Klebsiella pneumoniae (K. pneumo-
niae), Acinetobacter baumannii (A. baumannii), Pseudomonas
aeruginosa (P. aeruginosa), and Enterobacter spp. are the most
common microorganisms causing life-threatening infections.
Approximately 40% of hospital-acquired bacterial infections are
induced by these pathogens, and most are resistant to
commonly used antibiotics.[3] A joint program for development
of effective antimicrobial agents has been initiated by 18
European countries and Canada. Therefore, antibacterial agents
capable of fighting infections and spread of antibiotic resistance
are currently needed.[4]
The World Health Organization listed bacterial infections as a
foremost threat to global public health. Based on calculations,
multidrug-resistant bacterial pathogens are responsible for
[a] Dr. P. Ravichandiran, Prof. D. J. Yoo
Department of Life Science, Department of Energy Storage/Conversion
Engineering of Graduate School, and Hydrogen and Fuel Cell Research
Center, Chonbuk National University, Jeollabuk-do 54896, Republic of Korea
E-mail: ravichandru55@gmail.com
djyoo@jbnu.ac.kr
[b] Prof. M. Masłyk, Dr. M. Janeczko
Department of Molecular Biology, Faculty of Biotechnology and Environ-
mental Sciences, The John Paul II Catholic University of Lublin, ul.
Konstantynów 1i, 20-708 Lublin, Poland
Regarding the development of effective antimicrobial
agents, naphthoquinones have been the focus of extensive
research due to their varied functions and clinical applications.[5]
A number of natural products contain the naphthoquinone
moiety as the core structure. One example is vitamin K, which
plays a significant role in bone metabolism, vascular biology,
and regulation of blood coagulation in humans.[6] 1,4-Naphtho-
quinones consist of two ketone groups as vital chromophores
that can donate or accept electrons (redox) in a number of
biological systems. In recent studies, the inhibitory mechanism
of 1,4-naphthoquinone was shown to involve the production of
active oxygen species (ROS) by redox cycling, alkylation, or
intercalation in the DNA double helix of biomolecules.[7,8] 1,4-
Naphthoquinones and its related compounds are well-estab-
lished for irreversible complexation during ROS generation in
proteins, which leads to loss of protein function.[9] Due to these
properties, 1,4-naphthoquinone derivatives exhibit biological
[c] Dr. S. Sheet
Department of Forest Science and Technology, College of Agriculture and
Life Sciences, Chonbuk National University, 567 Baekje-daero, Deokjin-gu,
Jeonju-si 561-756, Jeollabuk-do, Republic of Korea
[d] Dr. D. Premnath
Department of Biotechnology, Karunya Institute of Technology and Science,
School of Agriculture and Biosciences, Karunya Nagar, Coimbatore 641114,
Tamil Nadu, India
[e] Prof. A. R. Kim
R&D Center for CANUTECH, Business Incubation Center, Department of
Bioenvironmental Chemistry, Chonbuk National University, Jeollabuk-do
54896, Republic of Korea.
[f] Prof. B.-H. Park
Department of Biochemistry, Chonbuk National University Medical School,
Jeollabuk-do 54896, Republic of Korea
[g] Prof. M.-K. Han
Department of Microbiology, Chonbuk National University Medical School,
Jeollabuk-do 54896, Republic of Korea
Supporting information for this article is available on the WWW under
https://doi.org/10.1002/open.201900077
©201x The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.
This is an open access article under the terms of the Creative Commons
Attribution Non-Commercial NoDerivs License, which permits use and dis-
tribution in any medium, provided the original work is properly cited, the
use is non-commercial and no modifications or adaptations are made.
properties
such
as
antimicrobial,[10]
antimalarial,[11]
antitubercular,[12] anticancer,[13] and trypanocidal[14] activities.
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Scheme 2. Synthesis of 2-(cyclohexylthio)-3-(phenylamino)naphthalene-1,4-
diones (5x–5aa).
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Recently, antimicrobial activities of 1,4-naphthoquinone
derivatives have attracted much attention.[15,16] Pingaew et al.[17]
reported the synthesis of novel 1,4-naphthoquinone-based
sulfonamides and antimicrobial and anticancer potentials
(compound I). Cardoso et al.[18] performed the synthesis and
antibacterial evaluation of sulfonamide-based lapachone deriv-
atives (compound II). Yıldırım et al.[19] reported the synthesis of
2,3-disubstituted-aryl-phenylthio-1,4-naphthoquinones as po-
tential antimicrobial agents (compound III). Tandon et al.[20,21]
described the synthesis and biological evaluation of several
new sulfanyl aminonaphthoquinones (compounds IV and V)
that displayed important antibacterial activity. Novais et al.[22]
also reported 1,4-naphthoquinone-based sulfanyl derivatives,
and their resistance toward Gram-negative bacteria in biofilms
was investigated (compound VI). However, the detailed toxicity
and action mechanism for these reported naphthoquinones in
humans are unknown to date (Figure 1).
Figure 1. Important nitrogen and sulfur containing naphthoquinones pos-
sesses interesting antimicrobial properties.
In earlier reports, we synthesized a panel of 1,4 naphthoqui-
none-based derivatives and evaluated their anticancer and
antibacterial properties.[23–26] Therefore, in the present study, the
synthesis and biological evaluation of
a new library of
arylamino-1,4-naphthoquinone derivatives grafted with S-
cyclohexane and aryl/aliphatic amide moieties as key counter-
parts was performed. The synthesized compounds (3–5aa)
were examined for in vitro antimicrobial property against a set
of Gram-positive and Gram-negative bacteria. The toxicity of
the selected compounds to human erythrocytes was tested
using a hemolysis assay. In addition, the potential antibacterial
activities of naphthoquinones using ROS generation, time-kill
kinetic study, apoptosis, and necrosis responses were deter-
mined.
2. Results and Discussion
2.1. Chemistry
A new series of 1,4-naphthoquinone-based compounds were
synthesized, and the synthetic routes for preparation of
compounds 3–5w and 5x–5aa are presented in Schemes 1 and
2. The parent molecule 3 was synthesized via the nucleophilic
substitution reaction between 2,3-dichloro-1,4-naphthoquinone
1 and 4-aminophenyl sulfone 2 using our previously reported
method.[27] Compound 4 was prepared by nucleophilic sub-
stitution of an equal molar concentration of compound 3 and
cyclohexanethiol in dry acetone. The same equivalent of
Scheme 1. Synthesis and substrate scope of 2-((4-((4-aminophenyl)sulfonyl)
phenyl)amino)-3-(cyclohexylthio)naphthalene-1,4-diones (3–5w).
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Table 1. In vitro antibacterial activity of 1,4-naphthoquinone derivatives (3–5aa) expressed as minimal inhibitory concentration.
MIC (μg/mL)
1
2
3
Compd.
S. aureus
E. coli
P. aeruginosa
S. bongori
K. pneumoniae
E faecalis
E. cloacae
C. albicans
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5
6
7
8
9
3
4
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
5l
5m
5n
5o
5p
5q
5r
5s
5t
5u
5v
5w
5x
5y
5z
150�0.23
>300
>300
>300
>300
>300
>300
>300
>300
>300
>300
>300
150�0.27
>300
>300
>300
>300
>300
>300
>300
>300
>300
>300
>300
>300
>300
>300
>300
>300
NA
NA
NA
NA
>300
150�0.13
>300
>300
150�0.84
>300
>300
>300
>300
>300
>300
>300
NA
>300
>300
>300
>300
NA
>300
>300
NA
>300
150�0.25
>300
>300
>300
>300
>300
>300
>300
NA
>300
150�0.27
NA
>300
>300
NA
>300
>300
>300
>300
>300
>300
NA
>300
>300
>300
>300
>300
NA
>300
>300
>300
>300
>300
NA
>300
>300
>300
NA
>300
NA
>300
>300
0.1�0.19
2�0.76
1.5�0.19
150�0.12
300�0.89
150�0.81
150�0.19
70�0.90
70�0.71
150�1.10
150�1.01
70�0.93
150�0.19
NA
150�0.70
70�1.20
NA
>300
>300
NA
NA
>300
>300
>300
150�0.21
300�0.99
>300
300�0.71
150�0.09
150�0.80
70�0.10
NA
>300
300�0.19
>300
>300
>300
>300
>300
>300
150�0.18
>300
>300
>300
>300
>300
>300
>300
>300
>300
>300
>300
>300
>300
300�0.55
>300
>300
>300
>300
>300
NA
150�0.11
150�0.08
300�0.41
>300
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150�1.10
150�0.91
150�0.14
>300
>300
NA
150�0.78
70�0.81
>300
>300
>300
>300
>300
>300
150�0.83
>300
>300
>300
>300
>300
>300
>300
150�0.70
>300
>300
>300
NA
NA
>300
>300
300�0.92
>300
>300
30�0.74
150�0.91
150�0.11
150�0.81
150�0.99
70�1.51
150�0.85
300�0.89
70�1.01
>300
>300
>300
NA
NA
300�0.28
300�0.20
>300
300�0.90
300�0.82
>300
300�0.69
300�0.12
>300
>300
>300
NA
150�0.12
70�0.80
300�0.11
>300
150�0.97
150�0.44
150�0.10
>300
>300
300�1.41
>300
>300
300�1.60
300�1.30
300�0.41
>300
>300
NA
5aa
>300
>300
NA
NA
1.9�1.10
NA
>300
NA
NA
>300
>300
NA
NA
NA
>300
NA
NA
Caspofungin
Amphotericin B
Chloramphenicol
Streptomycin
NA
7.8�0.91
NA
3.2�0.40
7.8�0.15
7.8�0.71
10.2�0.92
7.8�0.70
Results expressed in μg/mL; Concentration range used: 0.001–2 mg/mL; NA – No Activity; entries in boldface highlight MIC values <70 μg/mL.
trimethylamine was used as a base catalyst to move the
reaction forward. Under the 2-h reflux condition, the reaction
afforded compound 4 with a 97% yield. This reaction was also
assessed in water and other polar organic solvents such as
acetonitrile, methanol, ethanol, and dimethylformamide; how-
ever, the reaction was unsuccessful in terms of product yield
and formation of close byproducts. From compound 4, the final
desired compounds 5a–w were synthesized; compound 4 was
acylated with various lengths of substituted acid chlorides in
dry acetone for 30 min. Under the reflux condition, the reaction
generated the targeted molecules in moderate to good product
yield (65%–98%; Scheme 1). Compounds 5x–y in Scheme 2
were synthesized using our previously reported method.[23]
Compounds 5z–5aa were synthesized via approaches similar to
those used in Scheme 1. The products obtained had a moderate
to good percentage yield. A decrease in product yield (5i and
5l; 67% and 65%, respectively) was observed due to the
presence of bulky electron donating or electronegative func-
tional groups at the fourth position of the phenyl ring in the
molecule.
2.2. Biology
2.2.1. In Vitro Antibacterial Activity
Many of the 1,4-naphthoquinones have been well-documented
for synthetic utility and biologicals properties. However, specific
antibacterial targets of arylamino-1,4-naphthoquinones remain
to be evaluated in a biological study.[28–30]
The antibacterial activity of 29 different naphthoquinone
derivatives was screened against clinically important bacteria
(two strains of Gram-positive and five strains of Gram-negative
bacteria and one strain of pathogenic yeast) using caspofungin,
amphotericin B, chloramphenicol and streptomycin as standard
drugs (Table 1). MIC values were calculated using the broth
microdilution method according to the Clinical and Laboratory
Standards Institute (CLSI). The majority of the tested naphtho-
quinones showed low to good antibacterial activity ranging
from 30–300 μg/mL.
Among all the molecules (3–5aa) studied for antibacterial
potency, seven of naphthoquinones such as 5b, 5c, 5f, 5j, 5q,
5v, and 5y showed the notable activity against S. aureus, with
MIC values ranging from 30–70 μg/mL. Specifically, compound
5q showed potential antibacterial activity against S. aureus,
with an MIC of 30 μg/mL. Compounds 5c, 5d, 5g, 5i, 5j, 5u,
and 5v showed moderate antibacterial activity against P.
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aeruginosa, with MIC values ranging from 70–150 μg/mL. Similar
activity (70–150 μg/mL) was observed for compounds 5c, 5d,
5e, 5g, 5i, and 5u against S. bongori. Compared with the
parent molecules 3 and 4, the above-mentioned derivatives
exhibited better activity. However, the synthesized naphthoqui-
nones not showed any significant activity against the other
bacterial strains such as E. coli, K. pneumoniae, E. faecalis, and E.
cloacae, the yeast C. albicans.
Substitution of diaminobenzenesulfone and cyclohexane-
thiol moieties into compound 1 at the C-2 and C-3 positions
resulted in improved inhibitory activity. Introduction of an
amide group into compound 4 showed a good range of
antibacterial activity, although it was mainly contingent on
both the structural arrangement of the molecule and the
bacterial strain used in the experiment.[31]
Based on the results obtained, the structure-activity rela-
tionships (SARs) of the synthesized compounds (3–5aa) were
investigated. In summary, S-cyclohexane, sulfonated arylamine
moieties were found to be important for improved antibacterial
activity. Aryl amide group on the sulfone nucleus showed
encouraging antibacterial activity. Replacement of aryl amide
with aliphatic amide moiety showed the weaker antibacterial
activity. Electronegative groups (À F and À Br), and electron
withdrawing group (NO2) at position 2 and 3 of aryl amide
moiety were displayed promising activity. Similarly, furan-2-
carboxamide moiety also exhibited significant antibacterial
activity. Nevertheless, addition at position 4 on aryl amide did
not show any antibacterial activity. Hence, the present naph-
thoquinones established to be exceptional patterns for further
improvement as antibacterial candidates.
for all the molecules in this study, except for compound 5q, in
which a 2.33-fold higher concentration was used. The results
showed that all the compounds induced hemolysis in less than
3.4% of erythrocytes. In addition, vitamin K3 menadione (2-
methyl-1,4-naphthoquinone), a well-known naphthoquinone-
based antimicrobial drug, showed an MIC of 3 μg/mL and
induced hemolysis in 3.5% of erythrocytes (5 μg/mL).[32,33] In the
present study, all compounds at high dosage (70 μg/mL)
damaged approximately only 1–3.4% of human erythrocytes.
The toxicity results indicate that the present compounds are
less toxic to human erythrocytes.
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2.4. Detection of Naphthoquinone-Generated ROS
Improved ROS level is an important factor for the antibacterial
activity of 1,4-naphthoquinones.[34] Naphthoquinones are the
redox-active molecules that induce ROS after diffusion of the
bacterial cell membrane. Thus, three highly active representa-
tive naphthoquinones (5b, 5j, and 5q) with their respective
MIS values were selected for additional investigation of
antibacterial activity against S. aureus. The ROS generation
induced by naphthoquinone-treated cells was determined
using 2’,7’-dichlorofluorescein-diacetate (DCFH-DA), a non-fluo-
rescent intracellular ROS probe. During quantification of ROS
generation in cells, DCFH-DA oxidized into highly fluorescent
2’,7’-dichlorodihydrofluorescein (DCF) by the action of ROS.[35]
When S. aureus was treated with 5b, 5j, or 5q, the fluorescent
DCF dye was changed to DCF+ and showed good fluorescence
intensity due to the compound-induced intracellular ROS
formation (Figure 3). A population of DCF+ cells showed higher
fluorescence intensity for molecules 5b and 5j than 5q. The
compounds 5b and 5j exhibited similar fluorescence intensity
to the standard antibacterial drug streptomycin. This result
indicates that the antibacterial activity of 5b, 5j, and 5q
proceeded via oxidative stress of ROS. In addition, the redox
poteantial of these compounds were confirmed by cyclic
voltammetry study (see Figures S3–S5).
2.3. Hemolytic Activity in Human Erythrocytes
Toxicity is an important factor when a drug has potential as an
antimicrobial therapeutic agent. To verify the toxicity to human
erythrocytes, the most active compounds, 5b, 5c, 5f, 5j, 5q,
5v, and 5y, were evaluated for hemolytic activity; the results
are presented in Figure 2. The corresponding MICs were used
Figure 2. Hemolytic activity of compounds 5b, 5c, 5f, 5j, 5q, 5v, and 5y.
Tetracycline (TET) and chloramphenicol (CAM) were used as standard
controls. Triton X-100 (1%) and phosphate buffer solution (PBS) were used
as positive and negative controls, respectively. The concentration of
compound 5q used 2.33%×MIC value. The other compounds and anti-
biotics were tested in their respective MICs.
Figure 3. ROS detection of compounds 5b, 5j, and 5q against S. aureus.
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Figure 5. Apoptosis and necrosis determination was carried out by Annexin
V-FITC/PI double staining.
44.8, and 54.9% cell death in late apoptosis compared with
3.23% in control cells. In addition, 5j resulted in 48.9% of
necrosis cell death whereas, compound 5q displayed 37.2% of
early apoptosis cell death. Consequently, the apoptotic effect of
the compounds follows the order 5j>5b>5q. These results
indicate that the three naphthoquinone derivatives induce late
apoptosis in S. aureus at fixed concentrations.
Figure 4. a) Time-based bactericidal activity of compounds 5b, 5j, and 5q to
S. aureus. All the experiments carried out in triplicates and are given the
mean �S.D of CFU/mL. b) Images display S. aureus after 24-h treatment with
compounds 5b, 5j, and 5q.
2.5. Bactericidal Time-Kill Kinetic Study
2.7. In Silico Molecular Docking Study
The bactericidal activity of the most active naphthoquinones,
5b, 5j, and 5q, was also investigated using time-kill assays
against S. aureus at the respective MICs (70 μg/mL, 70 μg/mL,
and 30 μg/mL). The initial S. aureus count was 106.1 CFU/mL, and
the survival of bacteria started to decrease at 4-h and dropped
to 100 CFU/mL after 24-h. In contrast, compound 5q displayed
superior bactericidal kinetics compared with molecules 5b and
5j. The results indicate that all three compounds have time-
dependent bactericidal activity (Figure 4).
The mechanism binding naphthoquinones with S. aureus was
evaluated using a molecular docking approach. Three major
compounds, 5b, 5j, and 5q, were used for the study with
protein 5XEX (crystal structure of S. aureus PNPase catalytic
domain). In silico molecular docking results for the compounds
5b, 5j, and 5q showed molecular interactions with the
biologically active site of 5XEX with docking scores of
À 2.41 kcal/mol, À 3.13 kcal/mol, and À 3.34 kcal/mol, respec-
tively.
The predicted interactions of compound 5b show amide
NH, two oxygen of the sulfone group and one oxygen in
quinone moiety established hydrogen bonds with amino acid
residues of LYS A:251, ARG A:368, LYS A:463 and ASP A:477.
In addition, it is also noted, the presence of hydrophobic
interactions between the aromatic moieties of the molecule
and the non-polar amino acid residues such as SER A:403, SER
A:406, ASN A:404, GLY A:405, HIS A:372, ARG A:84, ARG A:87,
ARG A:88, ARG A:368, respectively (Figure 6).
2.6. Determination of Apoptosis and Necrosis Based on
Annexin V-FITC/PI Assay
Apoptosis is a process of programmed cell death and can be
inudced by certain ROS molecules. To investigate the apoptotic
mechanism of compounds 5b, 5j, and 5q, Annexin V-FITC/PI
assay was performed. The bacterial cells (S. aureus) were treated
with the corresponding MIC of compounds 5b, 5j, and 5q for
24-h and finally stained with Annexin-V and PI. The total
population of apoptotic cells was measured using flow
cytometry (Figure 5). Treatment of bacterial cells with 5b, 5j,
and 5q at 70, 70, and 90 μg/mL, respectively, resulted in 84.9,
In compound 5j two oxygen of the sulfone group and one
oxygen in quinone moiety forms hydrogen bonds with ARG
A:368, LYS A:463, LYS A:251. Further, hydrophobic interactions
were also presented between the aryl ketone and non-polar
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Experimental Section
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Chemistry
°
Melting points ( C) of all the naphthoquinone derivatives were
checked in an open capillary tube using a digital auto melting point
apparatus (Stuart, SMP10, Staffordshire, ST15 OSA, UK). All chem-
icals and solvents were purchased from Sigma-Aldrich, Alfa Aesar,
Korea and used directly without any further purification. Progress of
the reactions and purity of the products were checked by thin layer
chromatography on a TLC silica gel 60 F254 using the eluting
solvent ethyl acetate:hexane (1:1). All synthesized compounds
were preliminarily characterized by Fourier-transform infrared
spectroscopy analysis (Perkin-Elmer instrument with a resolution of
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31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
0.4 cmÀ 1) using the KBr pellet method. H NMR spectroscopy was
1
performed in DMSO-d6 (400 MHz, Jeol, JNM-ECZ400s) and 13C NMR
spectroscopy was performed in DMSO-d6 (100 MHz, Jeol, JNM-
ECZ400s) installed at the Center for University-Wide Research
Facilities (CURF) at Chonbuk National University (CBNU). The
coupling constants (J value) are reported in Hz. Elemental analysis
was carried out in a Flash 2000 Elemental Analyzer (Thermo Fisher
Scientific Inc.).
Figure 6. a) 2D ligand interaction of 5b with the amino acid active site of
5XEX. b) 3D docking of 5b (S=À 2.41 kcal/mol) in the active site of 5XEX. c)
Docking packing illustration of 5b with their suitable binding pockets of
5XEX.
amino acid residues. Similarly, compound 5q showed three
hydrogen bonding interactions with amide NH, amide oxygen
and quinone oxygen. Moreover, π-π stacking between sulfone
phenyl ring and naphthoquinone group with LYS A:479.
Hydrophobic interactions were also observed in aromatic
moieties of the molecule (Figures S1–S2).
Based on the results obtained from the docking study, the
experimental activity is comparable. The compound 5q showed
strong glide score of À 3.34 kcal/mol and better MIC of 30 μg/
mL. Besides compounds, 5b and 5j showed docking scores of
À 2.41 kcal/mol, À 3.13 kcal/mol with the MIC of 70 μg/mL
respectively. Therefore, the order of antibacterial activity
following this direction: 5q>5j>5b.
Synthesis of 2-((4-((4-aminophenyl)sulfonyl)phenyl)
amino)-3-chloronaphthalene-1,4-dione (3)
Compound
3 was synthesized using a previously reported
method[27] with minor modifications. In detail, a mixture of 2,3-
dichloro-1,4-naphthoquinone 1 (2.270 g, 10 mmol) and 4-amino-
phenyl sulfone 2 (2.048 g, 10 mmol) was added to double distilled
deionized water (900 mL) and the mixture was refluxed for 2 h. The
reaction mixture was cooled to room temperature. Deep red
precipitate that formed was isolated by vacuum filtration, washed
°
with hot water (500 mL) and dried at 50 C in a hot oven. The
obtained solid was purified by column chromatography using a
column silica gel 100–200 mesh (ethyl acetate:hexane, 1:2) to give
°
compound 3 as deep red crystals (88%); mp: 239–240 C; IR (KBr):
~
n=552, 697, 1105, 1300, 1560, 1678, 3242, 3364, 3451. 1H NMR
°
(400 MHz, DMSO-d6, 25 C, TMS): δ=6.14 (s, 2H), 6.62 (d, J=8.7 Hz,
Hence, these docking results indicate present groups of
naphthoquinone molecules are active candidates for further
clinical development.
2H), 7.19 (d, J=8.6 Hz, 2H), 7.54 (d, J=8.7 Hz, 2H), 7.72 (d, J=
8.6 Hz, 2H), 7.81 (dt, J=13.6 Hz & J=1.2 Hz, 1H), 7.87 (dt, J=7.4 Hz
& J=0.8 Hz, 1H), 8.03 (t, J=5.9 Hz, 2H), 9.52 (s, 1H) ppm. 13C NMR
(100 MHz, DMSO-d6): δ=112.9, 119.0, 122.2, 125.9, 126.1, 126.6,
126.7, 129.2, 130.4, 131.6, 133.5, 134.6, 137.0, 142.5, 143.1, 153.4,
176.9, 179.8 ppm. Anal. calcd for C22H15ClN2O4S (438): C, 60.21; H,
3.45; N, 6.38; S, 7.30. Found: C, 60.30; H, 3.36; N, 6.64; S, 7.31;
Beilstein test: Cl positive.[36]
3. Conclusions
In conclusion, the present investigation effectively demon-
strated the synthesis of desired naphthoquinone derivatives via
Michael-like addition, nucleophilic substitution, and acylation
reactions. The in vitro antimicrobial evaluation indicated that
insertion of arylamine, cyclohexanethiol, and amide moieties
Synthesis of 2-((4-((4-aminophenyl)sulfonyl)phenyl)
amino)-3-(cyclohexylthio)naphthalene-1,4-dione (4)
Compound 3 (2.19 g, 5 mmol) was mixed in a dry acetone (350 mL),
and cyclohexanethiol (0.611 mL, 5 mmol) was gradually added to
the mixture at room temperature. Trimethylamine (1 equiv.
0.469 mL) was added and the mixture was refluxed for 2 h. The
crude mixture was poured into crushed ice and the solid that
formed was filtered, washed with hot water (500 mL), dried in a hot
into the 1,4-naphthoquinone core could be
a beneficial
approach to enhance the antimicrobial activity. In contrast, at
least seven naphthoquinone derivatives showed distinguished
antibacterial activity against S. aureus at the collective anti-
bacterial level. In addition, three active compounds against S.
aureus were studied for their antibacterial mechanism via ROS
generation, time-kill kinetic assay, apoptosis and necrosis
responses. Studied compounds were toxic towards bacteria and
non-toxic to human erythrocytes. Thus, the results from this
study may aid in designing of clinically effective naphthoqui-
none-based antimicrobial agents for further development.
°
oven at 50 C and purified by silica column chromatography (100–
200 mesh, eluting solvents, ethyl acetate:hexane, 1:2) to give
°
~
compound 4, a maroon solid (97%); mp: 201–202 C; IR (KBr): n=
561, 693, 715, 1052, 1113, 1215, 1287, 1340, 1543, 1510, 1667, 3329,
3349, 3459. H NMR (400 MHz, DMSO-d6, 25 C, TMS): δ=1.01 (s,
5H), 1.40–1.49 (m, 5H), 2.89 (s, 1H), 6.13 (s, 2H), 6.60 (d, J=8.64 Hz,
2H), 7.11 (d, J=8.48 Hz, 2H), 7.50 (d, J=8.48 Hz, 2H), 7.69 (d, J=
8.60 Hz, 2H), 7.78 (t, J=7.40 Hz, 1H), 7.83 (t, J=7.32 Hz, 1H), 7.99 (d,
1
°
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Full Papers
J=7.28 Hz, 2H), 9.36 (s, 1H) ppm. 13C NMR (100 MHz, DMSO-d6): δ=
25.0, 25.4, 32.6, 44.6, 112.9, 121.0, 121.3, 126.1, 126.3, 126.6, 129.0,
130.8, 132.5, 133.3, 134.3, 135.9, 143.4, 144.8, 153.3, 179.6,
180.8 ppm. Anal. calcd for C28H26N2O4S2 (518): C, 64.84; H, 5.05; N,
5.40; S, 12.36 Found: C, 65.22; H, 5.40; N, 5.73; S, 12.70.
N-(4-((4-((3-(cyclohexylthio)-1,4-dioxo-1,4-dihydronaphthale-
n-2-yl)amino)phenyl)sulfonyl)phenyl)-3-(trifluoromethyl)
benzamide (5d)
1
2
3
4
5
6
7
8
9
°
~
Maroon solid (76%); mp: 156–157 C; IR (KBr): n=615, 720, 1082,
1115, 1258, 1312, 1658, 3279, 3431. 1H NMR (400 MHz, DMSO-d6,
°
25 C, TMS): δ=1.01 (s, 5H), 1.40–1.48 (m, 5H), 2.92 (s, 1H), 7.16 (d,
J=8.76 Hz, 2H), 7.79–7.85 (m, 5H), 7.93 (t, J=8.84 Hz, 2H), 7.97–7.99
(m, 2H), 8.00–8.02 (m, 3H), 8.24–8.28 (m, 2H), 9.401 (s, 1H), 10.82 (s,
1H) ppm. 13C NMR (100 MHz, DMSO-d6): δ=24.9, 25.3, 32.6, 44.6,
120.3, 120.8, 122.5, 124.3 (d, J=3.33 Hz), 125.2, 126.1, 126.2, 127.4,
128.0, 128.8 (q, J=31.91 Hz), 129.7, 130.8, 131.9, 132.5, 133.4, 133.7,
134.3, 135.1, 136.3, 143.1, 144.3, 144.5, 164.5, 179.5, 180.8 ppm. 19F
NMR (600 MHz, DMSO): δ=À 61.0 ppm. Anal. calcd for
C36H29F3N2O5S2 (690): C, 62.60; H, 4.23; N, 4.06; S, 9.28 Found: C,
62.81; H, 4.51; N, 3.80; S, 9.13.
General Procedure for the Preparation of Compounds 5a–5w
An equal stoichiometric ratio of compound 4 and various types of
acid chlorides were mixed in moisture dried acetone and the
reaction mixture was refluxed for 30 min. The obtained crude
mixture was poured into crushed ice and the obtained solid was
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
°
filtered through a vacuum. The solid was dried at 50 C and purified
by column chromatography (eluting solvent, ethyl acetate:hexane,
1:2) to obtain pure samples of 5a–5w.
N-(4-((4-((3-(cyclohexylthio)-1,4-dioxo-1,4-dihydronaphthale-
n-2-yl)amino)phenyl)sulfonyl)phenyl)benzamide (5a)
3,5-dichloro-N-(4-((4-((3-(cyclohexylthio)-1,4-dioxo-1,4-dihydro-
naphthalen-2-yl)amino)phenyl)sulfonyl)phenyl)benzamide (5e)
°
~
Violet solid (72%); mp: 156–157 C; IR (KBr): n=590, 720, 1090,
°
~
Maroon solid (98%); mp: 265–266 C; IR (KBr): n=696, 1038, 1209,
1122, 1235, 1350, 1523, 1667, 3319, 3451. 1H NMR (400 MHz, DMSO-
1
°
1251, 1663, 3179, 3429. H NMR (400 MHz, DMSO-d6, 25 C, TMS):
°
d6, 25 C, TMS): δ=1.01 (s, 5H), 1.39–1.48 (m, 5H), 2.92 (s, 1H), 7.15
δ=1.00 (s, 5H), 1.39–1.47 (m, 5H), 2.92 (s, 1H), 7.15 (d, J=8.80 Hz,
2H), 7.78–7.80 (m, 3H), 7.83 (dd, J=7.86 Hz & J=1.52 Hz, 1H), 7.86
(t, J=1.84 Hz, 1H), 7.91 (d, J=9.04 Hz, 2H), 7.96 (t, J=2.24 Hz, 3H),
7.99 (dd, J=7.34 Hz & J=1.48 Hz, 3H), 9.39 (s, 1H), 10.74 (s, 1H)
ppm. 13C NMR (100 MHz, DMSO-d6): δ=25.0, 25.3, 32.6, 44.6, 120.2,
120.8, 122.5, 126.1, 126.2, 126.5, 126.6, 127.4, 128.0, 130.8, 132.5,
133.4, 133.6, 134.6, 136.4, 137.4, 142.9, 144.3, 144.4, 163.1, 179.5,
180.8 ppm. Anal. calcd for C35H28Cl2N2O5S2 (691): C, 60.78; H, 4.08; N,
4.05; S, 9.27 Found: C, 60.42; H, 4.45; N, 4.02; S, 9.20. Beilstein test:
Cl positive.[36]
(d, J=8.68 Hz, 2H), 7.53 (t, J=7.68 Hz, 2H), 7.61 (t, J=6.96 Hz, 1H),
7.78–7.81 (m, 3H), 7.84 (d, J=7.04 Hz, 1H), 7.89 (d, J=8.76 Hz, 2H),
7.94 (d, J=7.48 Hz, 2H), 8.00 (t, J=8.52 Hz, 4H), 9.36 (s, 1H), 10.60
(s, 1H) ppm. 13C NMR (100 MHz, DMSO-d6): δ=24.9, 25.2, 32.6, 44.6,
120.0, 120.8, 122.4, 126.0, 126.2, 127.3, 127.7, 128.0, 128.3, 130.8,
131.9, 132.5, 133.3, 133.8, 134.2, 134.3, 135.9, 143.4, 144.2, 144.5,
166.0, 179.5, 180.8 ppm. Anal. calcd for C35H30N2O5S2 (622): C, 67.50;
H, 4.86; N, 4.50; S, 10.30 Found: C, 67.12; H, 5.10; N, 4.62; S, 10.65.
N-(4-((4-((3-(cyclohexylthio)-1,4-dioxo-1,4-dihydronaphthale-
n-2-yl)amino)phenyl)sulfonyl)phenyl)-2-fluorobenzamide (5b)
N-(4-((4-((3-(cyclohexylthio)-1,4-dioxo-1,4-dihydronaphthale-
n-2-yl)amino)phenyl)sulfonyl)phenyl)-3,5-dinitrobenzamide (5f)
°
~
Violet solid (82%); mp: 209–210 C; IR (KBr): n=570, 602, 1035,
1096, 1259, 1556, 1630, 3319, 3423. 1H NMR (400 MHz, DMSO-d6,
°
~
Violet solid (85%); mp: 278–279 C; IR (KBr): n=609, 721, 887, 982,
1
°
25 C, TMS): δ=1.01 (s, 5H), 1.39–1.49 (m, 5H), 2.90 (s, 1H), 7.15 (d,
1009, 1118, 1251, 1358, 1581, 1660, 3125, 3449. H NMR (400 MHz,
DMSO-d6, 25 C, TMS): δ=1.01 (s, 5H), 1.39–1.47 (m, 5H), 2.91 (s,
J=8.72 Hz, 2H), 7.34 (q, J=10.56 Hz, 2H), 7.57–7.62 (m, 1H), 7.66 (t,
J=8.84 Hz, 1H), 7.77–7.81 (m, 3H), 7.84 (d, J=6.20 Hz, 1H), 7.91 (d,
J=3.52 Hz, 4H), 8.00 (d, J=7.76 Hz, 2H), 9.40 (s, 1H), 10.83 (s, 1H)
ppm. 13C NMR (100 MHz, DMSO-d6): δ=25.0, 25.3, 32.6, 44.6, 116.1,
116.3, 119.6, 120.9, 122.4, 124.6, 124.6, 126.1, 126.3, 127.3, 128.2,
129.8, 130.9, 132.5, 133.4, 133.7, 134.3, 136.2, 143.0, 144.2, 144.4,
158.8 (d, J=247.95 Hz), 163.3, 179.6, 180.9 ppm. 19F NMR (600 MHz,
DMSO): δ=À 114.4 ppm. Anal. calcd for C35H29FN2O5S2 (640): C,
65.61; H, 4.56; N, 4.37; S, 10.01 Found: C, 65.32; H, 4.70; N, 4.62; S,
10.35.
°
1H), 7.16 (d, J=8.68 Hz, 2H), 7.77–7.83 (m, 4H), 7.96 (t, J=4.84 Hz,
3H), 8.00 (t, J=8.88 Hz, 3H), 9.00 (t, J=1.88 Hz, 1H), 9.14 (d, J=
1.92 Hz, 2H), 9.39 (s, 1H), 11.15 (s, 1H) ppm. 13C NMR (100 MHz,
DMSO-d6): δ=25.0, 25.3, 32.6, 44.6, 120.6, 120.8, 121.4, 122.6, 126.1,
126.2, 127.4, 128.1, 130.8, 132.5, 133.4, 133.5, 134.3, 136.8, 136.9,
142.6, 144.3, 144.4, 148.0, 161.8, 179.5, 180.8 ppm. Anal. calcd for
C35H28N4O9S2 (712): C, 58.98; H, 3.96; N, 7.86; S, 9.00 Found: C, 58.61;
H, 4.25; N, 7.50; S, 9.27.
N-(4-((4-((3-(cyclohexylthio)-1,4-dioxo-1,4-dihydronaphthale-
n-2-yl)amino)phenyl)sulfonyl)phenyl)-3-nitrobenzamide (5g)
2-bromo-N-(4-((4-((3-(cyclohexylthio)-1,4-dioxo-1,4-dihydronap-
hthalen-2-yl)amino)phenyl)sulfonyl)phenyl)benzamide (5c)
°
~
Blue violet solid (89%); mp: 165–166 C; IR (KBr): n=598, 612, 890,
1102, 1237, 1599, 1665, 3115, 3441. 1H NMR (400 MHz, DMSO-d6,
°
~
Maroon solid (77%); mp: 244–245 C; IR (KBr): n=540, 653, 758, 987,
1024, 1096, 1213, 1358, 1478, 1548, 1661, 3329, 3438. 1H NMR
°
25 C, TMS): δ=1.01 (s, 5H), 1.39–1.47 (m, 5H), 2.91 (s, 1H), 7.15 (d,
°
(400 MHz, DMSO-d6, 25 C, TMS): δ=1.00 (s, 5H), 1.39–1.48 (m, 5H),
J=8.68 Hz, 2H), 7.79–7.83 (m, 4H), 7.85 (d, J=7.80 Hz, 1H), 7.93 (d,
J=8.80 Hz, 2H), 8.00 (t, J=9.12 Hz, 4H), 8.38 (d, J=7.84 Hz, 1H),
8.44 (d, J=8.28 Hz, 1H), 8.77 (s, 1H), 9.40 (s, 1H), 10.92 (s, 1H) ppm.
13C NMR (100 MHz, DMSO-d6): δ=24.9, 25.3, 32.6, 44.6, 120.3, 120.8,
122.5, 126.1, 126.2, 126.5, 127.4, 128.1, 130.2, 130.8, 132.5, 133.4,
133.6, 134.3, 134.3, 135.6, 136.4, 143.0, 144.3, 144.4, 147.6, 163.8,
179.5, 180.8 ppm. Anal. calcd for C35H29N3O7S2 (667): C, 62.95; H,
4.38; N, 6.29; S, 9.60 Found: C, 63.23; H, 4.20; N, 5.90; S, 9.62.
2.88 (s, 1H), 7.15 (d, J=8.68 Hz, 2H), 7.43 (t, J=7.56 Hz, 1H), 7.50 (t,
J=7.36 Hz, 1H), 7.56 (d, J=7.56 Hz, 1H), 7.72 (d, J=7.88 Hz, 1H),
7.78–7.80 (m, 3H), 7.84 (d, J=6.88 Hz, 1H), 7.91 (s, 4H), 8.00 (d, J=
7.40 Hz, 2H), 9.41 (s, 1H), 10.91 (s, 1H) ppm. 13C NMR (100 MHz,
DMSO-d6): δ=25.0, 25.3, 32.6, 44.6, 118.8, 119.5, 120.9, 122.3, 126.1,
126.2, 127.3, 127.7, 128.2, 128.8, 130.8, 131.4, 132.5, 132.7, 133.3,
133.7, 134.3, 136.2, 138.4, 143.0, 144.1, 144.3, 166.2, 179.5,
180.8 ppm. Anal. calcd for C35H29BrN2O5S2 (700): C, 59.91; H, 4.17; N,
3.99; S, 9.14 Found: C, 60.21; H, 4.54; N, 4.32; S, 8.82.
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N-(4-((4-((3-(cyclohexylthio)-1,4-dioxo-1,4-dihydronaphthale-
n-2-yl)amino)phenyl)sulfonyl)
phenyl)-4-methyl-3-nitrobenzamide (5h)
4-(tert-butyl)-N-(4-((4-((3-(cyclohexylthio)-1,4-dioxo-1,4-dihydro-
naphthalen-2-yl)amino)phenyl)sulfonyl)phenyl)benzamide (5l)
1
2
3
4
5
6
7
8
9
°
~
Blue violet solid (65%); mp: 163–164 C; IR (KBr): n=781, 889,
1
°
°
11029, 1351, 1668, 3325, 3402. H NMR (400 MHz, DMSO-d6, 25 C,
~
Maroon solid (98%); mp: 268–269 C; IR (KBr): n=912, 1189, 1217,
1348, 1492, 1572, 1662, 3105, 3402. 1H NMR (400 MHz, DMSO-d6,
TMS): δ=1.00 (s, 5H), 1.29 (s, 9H), 1.38–1.47 (m, 5H), 2.91 (s, 1H),
7.15 (d, J=8.68 Hz, 2H), 7.54 (d, J=8.40 Hz, 2H), 7.78–7.84 (m, 4H),
7.87 (d, J=5.32 Hz, 2H), 7.89 (d, J=5.68 Hz, 2H), 7.98–8.02 (m, 4H),
9.39 (s, 1H), 10.54 (s, 1H) ppm. 13C NMR (100 MHz, DMSO-d6): δ=
25.0, 25.3, 30.8, 32.6, 34.6, 44.6, 119.9, 120.9, 122.4, 125.2, 126.1,
126.2, 127.3, 127.6, 128.0, 130.8, 131.6, 132.5, 133.3, 133.9, 134.3,
135.7, 143.6, 144.2, 144.5, 154.9, 165.9, 179.5, 180.8 ppm. Anal. calcd
for C39H38N2O5S2 (678): C, 69.00; H, 5.64; N, 4.13; S, 9.45 Found: C,
69.08; H, 5.45; N, 4.39; S, 9.42.
°
25 C, TMS): δ=1.01 (s, 5H), 1.39–1.48 (m, 5H), 2.59 (s, 3H), 2.92 (s,
1H), 7.15 (d, J=8.64 Hz, 2H), 7.68 (d, J=8.08 Hz, 1H), 7.79 (d, J=
8.64 Hz, 3H), 7.83 (d, J=7.16 Hz, 1H), 7.91 (d, J=8.80 Hz, 2H), 7.99
(s, 3H), 8.01 (d, J=3.28 Hz, 1H), 8.19 (d, J=8.12 Hz, 1H), 8.55 (s, 1H),
9.36 (s, 1H), 10.78 (s, 1H) ppm. 13C NMR (100 MHz, DMSO-d6): δ=
19.5, 24.9, 25.3, 32.6, 44.6, 120.2, 120.8, 122.5, 123.6, 126.0, 126.2,
127.3, 128.0, 130.8, 132.1, 132.5, 133.0, 133.1, 133.3, 133.7, 134.3,
136.3, 136.6, 143.0, 144.3, 144.5, 148.7, 163.6, 179.5, 180.8 ppm.
Anal. calcd for C36H31N3O7S2 (681): C, 63.42; H, 4.58; N, 6.16; S, 9.41
Found: C, 63.11; H, 4.67; N, 6.15; S, 9.37.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
N-(4-((4-((3-(cyclohexylthio)-1,4-dioxo-1,4-dihydronaphthale-
n-2-yl)amino)phenyl)sulfonyl)phenyl)-4-fluorobenzamide (5m)
4-chloro-N-(4-((4-((3-(cyclohexylthio)-1,4-dioxo-1,4-dihydronap-
hthalen-2-yl)amino)phenyl)sulfonyl)phenyl)-3-nitrobenzamide
(5i)
°
~
Blue violet solid (97%); mp: 242–243 C; IR (KBr): n=682, 714, 882,
1
1108, 1251, 1327, 1482, 1509, 1661, 3314, 3342. H NMR (400 MHz,
DMSO-d6, 25 C, TMS): δ=1.00 (s, 5H), 1.38–1.47 (m, 5H), 2.91 (s,
°
1H), 7.15 (d, J=8.76 Hz, 2H), 7.37 (t, J=8.88 Hz, 2H), 7.78–7.80 (m,
3H), 7.83 (dd, J=7.36 Hz & J=1.40 Hz, 1H), 7.90 (d, J=8.88 Hz, 2H),
7.98–8.04 (m, 6H), 9.39 (s, 1H), 10.63 (s, 1H) ppm. 13C NMR (100 MHz,
DMSO-d6): δ=25.0, 25.3, 32.6, 44.6, 115.3, 115.5, 120.1, 120.9, 122.4,
126.1, 126.2, 127.4, 128.0, 130.5, 130.6, 132.5, 133.4, 133.8, 134.3,
136.0, 143.4, 144.2, 144.5, 164.2 (d, J=248.53 Hz), 164.9, 179.5,
180.8 ppm. 19F NMR (600 MHz, DMSO): δ=À 107.8 ppm. Anal. calcd
for C35H29FN2O5S2 (640): C, 65.61; H, 4.56; N, 4.37; S, 10.01 Found: C,
65.25; H, 4.62; N, 4.30; S, 10.00.
°
~
Light maroon solid (67%); mp: 169–170 C; IR (KBr): n=698, 751,
1
890, 1101, 1216, 1309, 1412, 1508, 1598, 1660, 3009, 3389. H NMR
(400 MHz, DMSO-d6, 25 C, TMS): δ=1.01 (s, 5H), 1.39–1.49 (m, 5H),
°
2.91 (s, 1H), 7.15 (d, J=7.68 Hz, 2H), 7.78–7.85 (m, 3H), 7.93 (d, J=
8.00 Hz, 2H), 7.96–8.00 (m, 6H), 8.24 (d, J=8.56 Hz, 1H), 8.62 (s, 1H),
9.39 (s, 1H), 10.88 (s, 1H) ppm. 13C NMR (100 MHz, DMSO-d6): δ=
25.0, 25.3, 32.6, 44.7, 120.3, 120.9, 122.5, 125.0, 126.1, 126.3, 127.4,
128.1, 128.5, 130.8, 132.0, 132.5, 132.9, 133.4, 133.6, 134.2, 134.3,
136.6, 142.8, 144.3, 144.4, 147.3, 163.0, 179.6, 180.9 ppm. Anal. calcd
for C35H28ClN3O7S2 (701): C, 59.87; H, 4.02; N, 5.98; S, 9.13 Found: C,
59.80; H, 4.32; N, 6.02; S, 8.98. Beilstein test: Cl positive.[36]
4-chloro-N-(4-((4-((3-(cyclohexylthio)-1,4-dioxo-1,4-dihydronap-
hthalen-2-yl)amino)phenyl)sulfonyl)phenyl)benzamide (5n)
N-(4-((4-((3-(cyclohexylthio)-1,4-dioxo-1,4-dihydronaphthale-
n-2-yl)amino)phenyl)sulfonyl)phenyl)-4-methoxybenzamide (5j)
°
~
Blue violet solid (98%); mp: 229–230 C; IR (KBr): n=664, 712, 882,
941, 1108, 1201, 1352, 1482, 1527, 1662, 3142, 3340. 1H NMR
°
(400 MHz, DMSO-d6, 25 C, TMS): δ=1.00 (s, 5H), 1.38–1.47 (m, 5H),
°
~
Blue violet solid (90%); mp: 250–251 C; IR (KBr): n=718, 887, 925,
1108, 1251, 1392, 1582, 1668, 3142, 3392. 1H NMR (400 MHz, DMSO-
2.91 (s, 1H), 7.15 (d, J=8.52 Hz, 2H), 7.55 (d, J=8.56 Hz, 1H), 7.61 (d,
J=8.56 Hz, 2H), 7.78–7.84 (m, 4H), 7.91 (t, J=3.68 Hz, 2H), 7.95 (d,
J=6.76 Hz, 1H), 7.98 (d, J=1.84 Hz, 3H), 8.00 (d, J=1.84 Hz, 1H),
9.39 (s, 1H), 10.67 (s, 1H) ppm. 13C NMR (100 MHz, DMSO-d6): δ=
25.5, 25.9, 33.1, 45.2, 120.7, 121.4, 126.6, 126.8, 127.9, 128.6, 129.0,
129.2, 130.3, 131.6, 133.1, 133.5, 133.9, 134.3, 134.9, 136.6, 137.4,
143.8, 144.8, 145.0, 165.5, 180.1, 181.4 ppm. Anal. calcd for
C35H29ClN2O5S2 (657): C, 63.96; H, 4.45; N, 4.26; S, 9.76 Found: C,
64.05; H, 4.32; N, 4.27; S, 9.91. Beilstein test: Cl positive.[36]
°
d6, 25 C, TMS): δ=1.00 (s, 5H), 1.38–1.47 (m, 5H), 2.37 (s, 3H), 2.91
(s, 1H), 7.15 (d, J=8.60 Hz, 2H), 7.33 (d, J=8.00 Hz, 2H), 7.76–7.82
(m, 4H), 7.87 (t, J=9.68 Hz, 4H), 8.00 (t, J=8.64 Hz, 4H), 8.39 (s, 1H),
10.52 (s, 1H) ppm. 13C NMR (100 MHz, DMSO-d6): δ=21.0, 25.0, 25.3,
32.6, 44.6, 120.0, 120.9, 122.4, 126.1, 126.3, 127.3, 127.8, 128.0,
128.9, 130.8, 131.4, 132.5, 133.4, 133.8, 134.3, 135.8, 142.1. 143.6,
144.2, 144.5, 165.8, 179.6, 180.8 ppm. Anal. calcd for C36H32N2O6S2
(652): C, 66.24; H, 4.94; N, 4.29; S, 9.82 Found: C, 66.09; H, 4.89; N,
3.97; S, 9.80.
4-bromo-N-(4-((4-((3-(cyclohexylthio)-1,4-dioxo-1,4-dihydronap-
hthalen-2-yl)amino)phenyl)sulfonyl)phenyl)benzamide (5o)
N-(4-((4-((3-(cyclohexylthio)-1,4-dioxo-1,4-dihydronaphthale-
n-2-yl)amino)phenyl)sulfonyl)phenyl)-4-methylbenzamide (5k)
°
~
Blue violet solid (92%); mp: 241–242 C; IR (KBr): n=754, 821, 994,
1108, 1251, 1415, 1598, 1665, 3214, 3418. 1H NMR (400 MHz, DMSO-
°
~
Blue violet solid (87%); mp: 256–257 C; IR (KBr): n=852, 918, 1119,
1282, 1352, 1480, 1501, 1648, 3109, 3402. 1H NMR (400 MHz, DMSO-
°
d6, 25 C, TMS): δ=1.00 (s, 5H), 1.38–1.47 (m, 5H), 2.91 (s, 1H), 7.15
(d, J=8.64 Hz, 2H), 7.75 (d, J=8.56 Hz, 2H), 7.78–7.84 (m, 4H), 7.90
(d, J=9.92 Hz, 4H), 7.99 (d, J=8.08 Hz, 4H), 9.39 (s, 1H), 10.66 (s, 1H)
ppm. 13C NMR (100 MHz, DMSO-d6): δ=25.0, 25.3, 32.6, 44.6, 120.1,
120.8, 122.4, 125.8, 126.1, 126.2, 127.3, 128.0, 129.9, 130.8, 131.4,
132.5, 133.3, 133.4, 133.7, 134.3, 136.1, 143.3, 144.2, 144.5, 165.0,
179.5, 180.8 ppm. Anal. calcd for C35H29BrN2O5S2 (701): C, 59.91; H,
4.17; N, 3.99; S, 9.14 Found: C, 59.97; H, 4.32; N, 4.15; S, 8.99.
°
d6, 25 C, TMS): δ=1.00 (s, 5H), 1.38–1.47 (m, 5H), 2.91 (s, 1H), 3.83
(s, 3H), 7.06 (d, J=7.40 Hz, 2H), 7.15 (d, J=7.68 Hz, 2H), 7.76–7.84
(m, 4H), 7.88 (d, J=7.76 Hz, 2H), 7.95 (d, J=7.52 Hz, 2H), 7.98–8.01
(m, 4H), 9.39 (s, 1H), 10.45 (s, 1H) ppm. 13C NMR (100 MHz, DMSO-
d6): δ=25.0, 25.3, 32.6, 44.6, 55.4, 113.6, 119.9, 120.9, 122.3, 126.1,
126.2, 126.2, 127.3, 128.0, 129.8, 130.8, 132.5, 133.4, 133.9, 134.3,
135.6, 143.7, 144.2, 144.5, 162.2, 165.3, 179.5, 180.8 ppm. Anal. calcd
for C36H32N2O5S2 (636): C, 67.90; H, 5.07; N, 4.40; S, 10.07 Found: C,
67.52; H, 4.98; N, 4.30; S, 10.22.
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N-(4-((4-((3-(cyclohexylthio)-1,4-dioxo-1,4-dihydronaphthale-
n-2-yl)amino)phenyl)sulfonyl)phenyl)-4-nitrobenzamide (5p)
N-(4-((4-((3-(cyclohexylthio)-1,4-dioxo-1,4-dihydronaphthale-
n-2-yl)amino)phenyl)sulfonyl)phenyl)-1-naphthamide (5t)
1
2
3
4
5
6
7
8
9
°
°
~
~
Blue violet solid (83%); mp: 169–170 C; IR (KBr): n=754, 821, 994,
Maroon solid (90%); mp: 242–243 C; IR (KBr): n=618, 722, 920,
1108, 1251, 1415, 1598, 1665, 3214, 3418. 1H NMR (400 MHz, DMSO-
1109, 1251, 1366, 1501, 1660, 3142, 3409. 1H NMR (400 MHz, DMSO-
°
°
d6, 25 C, TMS): δ=1.00 (s, 5H), 1.38–1.47 (m, 5H), 2.91 (s, 1H), 7.15
d6, 25 C, TMS): δ=1.02 (s, 5H), 1.40–1.49 (m, 5H), 2.91 (s, 1H), 7.16
(d, J=8.60 Hz, 2H), 7.76–7.83 (m, 4H), 7.92 (d, J=8.72 Hz, 2H), 7.99
(t, J=8.68 Hz, 4H), 8.16 (d, J=8.60 Hz, 2H), 8.36 (d, J=8.56 Hz, 2H),
9.39 (s, 1H), 10.91 (s, 1H) ppm. 13C NMR (100 MHz, DMSO-d6): δ=
25.0, 25.3, 32.6, 44.6, 120.3, 120.8, 122.5, 123.5, 126.1, 126.2, 127.4,
128.1, 129.3, 130.8, 132.5, 133.4, 133.6, 134.3, 136.5, 139.9, 143.0,
144.3, 144.4, 149.3, 164.4, 179.5, 180.8 ppm. Anal. calcd for
C35H29N3O7S2 (667): C, 62.95; H, 4.38; N, 6.29; S, 9.60 Found: C, 62.89;
H, 4.39; N, 6.48; S, 9.92.
(d, J=8.56 Hz, 2H), 7.57–7.63 (m, 3H), 7.75–7.83 (m, 5H), 7.93 (d, J=
8.60 Hz, 2H), 8.01 (t, J=8.72 Hz, 5H), 8.09 (d, J=8.24 Hz, 1H), 8.14
(dd, J=6.20 Hz & J=3.44 Hz, 1H), 9.41 (s, 1H), 10.99 (s, 1H) ppm. 13
C
NMR (100 MHz, DMSO-d6): δ=25.0, 25.3, 32.6, 44.6, 119.7, 120.9,
122.3, 124.8, 124.9, 125.7, 126.1, 126.3, 126.4, 127.1, 127.3, 128.2,
128.3, 129.4, 130.5, 130.8, 132.5, 133.1, 133.4, 133.9, 133.9, 134.3,
136.0, 143.5, 144.2, 144.5, 167.7, 179.6, 180.9 ppm. Anal. calcd for
C39H32N2O5S2 (672): C, 69.62; H, 4.79; N, 4.16; S, 9.53 Found: C, 69.50;
H, 4.71; N, 4.18; S, 9.60.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
N-(4-((4-((3-(cyclohexylthio)-1,4-dioxo-1,4-dihydronaphthale-
n-2-yl)amino)phenyl)sulfonyl)phenyl)furan-2-carboxamide (5q)
N-(4-((4-((3-(cyclohexylthio)-1,4-dioxo-1,4-dihydronaphthale-
n-2-yl)amino)phenyl)sulfonyl)phenyl)propionamide (5u)
°
~
Blue violet solid (94%); mp: 212–213 C; IR (KBr): n=653, 714, 833,
1011, 1254, 1385, 1522, 1662, 3145, 3421. 1H NMR (400 MHz, DMSO-
Blue violet solid (79%); mp: 153–154 C; IR (KBr): n=930, 1108,
°
~
d6, 25 C, TMS): δ=1.00 (s, 5H), 1.38–1.48 (m, 5H), 2.90 (s, 1H), 6.71
1251, 1420, 1588, 1662, 3112, 3425. 1H NMR (400 MHz, DMSO-d6,
°
°
(dd, J=3.48 Hz & J=1.76 Hz, 1H), 7.14 (d, J=8.76 Hz, 2H), 7.38 (d,
J=3.56 Hz, 1H), 7.77–7.81 (m, 3H), 7.83 (dd, J=7.40 Hz & J=
1.32 Hz, 1H), 7.87 (d, J=8.84 Hz, 2H), 7.95–8.00 (m, 5H), 9.36 (s, 1H),
10.53 (s, 1H) ppm. 13C NMR (100 MHz, DMSO-d6): δ=24.9, 25.2, 32.5,
44.6, 112.2, 115.6, 120.0, 120.8, 122.4, 126.0, 126.2, 127.3, 127.9,
130.8, 132.5, 133.3, 133.7, 134.3, 136.0, 142.9, 144.2, 144.4, 146.1,
146.8, 156.3, 179.5, 180.8 ppm. Anal. calcd for C33H28N2O6S2 (612): C,
64.69; H, 4.61; N, 4.57; S, 10.47 Found: C, 64.38; H, 4.65; N, 4.58; S,
10.20.
25 C, TMS): δ=1.00 (s, 5H), 1.06 (t, J=7.48 Hz, 3H), 1.39–1.47 (m,
5H), 2.34 (q, J=7.60 Hz, 2H), 2.90 (s, 1H), 7.13 (d, J=8.72 Hz, 2H),
7.76 (d, J=8.60 Hz, 3H), 7.80 (d, J=10.28 Hz, 4H), 7.83–7.85 (m, 1H),
7.99 (dd, J=7.50 Hz & J=1.0 Hz, 2H), 9.35 (s, 1H), 10.25 (s, 1H) ppm.
13C NMR (100 MHz, DMSO-d6): δ=9.3, 24.9, 25.2, 29.5, 32.5, 44.6,
118.7, 120.8, 122.3, 126.0, 126.2, 127.2, 128.1, 130.8, 132.5, 133.3,
133.9, 134.3, 135.1, 143.5, 144.1, 144.5, 172.6, 179.5, 180.8 ppm.
Anal. calcd for C31H30N2O5S2 (574): C, 64.79; H, 5.26; N, 4.87; S, 11.16
Found: C, 64.53; H, 5.23; N, 4.98; S, 11.51.
N-(4-((4-((3-(cyclohexylthio)-1,4-dioxo-1,4-dihydronaphthale-
n-2-yl)amino)phenyl)sulfonyl)phenyl)thiophene-2-carboxamide
(5r)
N-(4-((4-((3-(cyclohexylthio)-1,4-dioxo-1,4-dihydronaphthale-
n-2-yl)amino)phenyl)sulfonyl)phenyl)acetamide (5v)
°
~
Blue violet solid (96%); mp: 159–160 C; IR (KBr): n=1129, 1358,
1
°
°
1471, 1556, 1658, 3142, 3408. H NMR (400 MHz, DMSO-d6, 25 C,
~
Blue violet solid (88%); mp: 224–225 C; IR (KBr): n=915, 1105,
1248, 1355, 1508, 1650, 3140, 3411. 1H NMR (400 MHz, DMSO-d6,
TMS): δ=0.99 (s, 5H), 1.39–1.46 (m, 5H), 2.06 (s, 3H), 2.89 (s, 1H),
7.14 (d, J=8.64 Hz, 2H), 7.76 (d, J=8.72 Hz, 5H), 7.82 (d, J=8.64 Hz,
3H), 7.99 (d, J=7.36 Hz, 2H), 9.38 (s, 1H), 10.34 (s, 1H) ppm. 13C NMR
(100 MHz, DMSO-d6): δ=24.6, 25.5, 25.9, 33.1, 45.2, 119.3, 121.4,
122.9, 126.6, 126.8, 127.8, 128.7, 131.4, 133.0, 133.9, 134.4, 134.9,
135.8, 144.0, 144.7, 145.0, 169.6, 180.1, 181.4 ppm. Anal. calcd for
C30H28N2O5S2 (560): C, 64.26; H, 5.03; N, 5.00; S, 11.44 Found: C,
64.49; H, 5.01; N, 5.24; S, 11.80.
°
25 C, TMS): δ=1.00 (s, 5H), 1.38–1.46 (m, 5H), 2.90 (s, 1H), 7.15 (d,
J=8.68 Hz, 2H), 7.23 (t, J=4.60 Hz, 1H), 7.78–7.84 (m, 4H), 7.89 (d,
J=8.68 Hz, 3H), 7.95–8.00 (m, 4H), 8.05 (d, J=3.72 Hz, 1H), 9.39 (s,
1H), 10.56 (s, 1H) ppm. 13C NMR (100 MHz, DMSO-d6): δ=24.9, 25.3,
32.6, 44.6, 120.0, 120.9, 122.4, 126.1, 126.2, 127.4, 128.1, 128.1,
129.9, 130.8, 132.5, 132.7, 133.4, 133.7, 134.3, 135.9, 139.1, 143.1,
144.2, 144.4, 160.2, 179.5, 180.8 ppm. Anal. calcd for C33H28N2O5S3
(628): C, 63.04; H, 4.49; N, 4.46; S, 15.30 Found: C, 63.01; H, 4.54; N,
4.80; S, 15.13.
2,2-dichloro-N-(4-((4-((3-(cyclohexylthio)-1,4-dioxo-1,4-dihydro-
naphthalen-2-yl)amino)phenyl)sulfonyl)phenyl)acetamide (5w)
2-(4-chlorophenyl)-N-(4-((4-((3-(cyclohexylthio)-1,4-dioxo-1,4-di-
hydronaphthalen-2-yl)amino)phenyl)sulfonyl)phenyl)acetamide
(5s)
°
~
Maroon solid (87%); mp: 154–155 C; IR (KBr): n=971, 1125, 1389,
1
°
1582, 1668, 3125, 3420. H NMR (400 MHz, DMSO-d6, 25 C, TMS):
δ=0.99 (s, 5H), 1.38–1.47 (m, 5H), 2.89 (s, 1H), 6.59 (s, 1H), 7.14 (d,
J=8.72 Hz, 2H), 7.76–7.79 (m, 5H), 7.84 (d, J=7.28 Hz, 1H), 7.91 (d,
J=8.80 Hz, 2H), 8.00 (dd, J=7.28 Hz & J=1.30 Hz, 2H), 9.37 (s, 1H),
11.02 (s, 1H) ppm. 13C NMR (100 MHz, DMSO-d6): δ=24.9, 25.2, 32.5,
44.6, 67.0, 119.9, 120.8, 122.5, 126.0, 126.2, 127.3, 128.3, 130.8,
132.5, 133.3, 133.4, 134.3, 137.1, 141.7, 144.3, 144.3, 162.2, 179.5,
180.8 ppm. Anal. calcd for C30H26Cl2N2O5S2 (629): C, 57.23; H, 4.16; N,
4.45; S, 10.19 Found: C, 57.42; H, 4.53; N, 4.81; S, 10.15. Beilstein test:
Cl positive.[36]
°
~
Maroon solid (88%); mp: 147–148 C; IR (KBr): n=911, 1189, 1345,
1
°
1508, 1612, 1660, 3142, 3400. H NMR (400 MHz, DMSO-d6, 25 C,
TMS): δ=0.99 (s, 5H), 1.37–1.44 (m, 5H), 2.88 (s, 1H), 3.68 (s, 2H),
7.13 (d, J=8.76 Hz, 2H), 7.34 (q, J=8.48 Hz, 4H), 7.74 (s, 1H), 7.77 (d,
J=8.52 Hz, 3H), 7.81 (t, J=2.12 Hz, 1H), 7.83 (s, 2H), 7.85 (d, J=
1.52 Hz, 1H), 7.98 (d, J=1.32 Hz, 1H), 8.00 (d, J=1.24 Hz, 1H), 9.35
(s, 1H), 10.56 (s, 1H) ppm. 13C NMR (100 MHz, DMSO-d6): δ=24.9,
25.2, 32.5, 42.3, 44.6, 119.0, 120.8, 122.3, 126.0, 126.2, 127.2, 128.1,
128.1, 130.8, 130.9, 131.3, 132.5, 133.3, 133.8, 134.3, 134.3, 135.6,
143.2, 144.1, 144.4, 169.4, 179.5, 180.8 ppm. Anal. calcd for
C36H31ClN2O5S2 (671): C, 64.42; H, 4.66; N, 4.17; S, 9.55 Found: C,
64.20; H, 4.61; N, 4.47; S, 9.60. Beilstein test: Cl positive.[36]
General Procedure for the Synthesis of Compounds 5x–5y
2,3-Dichloro-1,4-naphthoquinone 1 (2.270 g, 10 mmol) and aryl
amines 2a–b (10 mmol) were added to double distilled water
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(800 mL) and the reaction mixture was refluxed for 2–3 h. The
mixture was cooled to room temperature and the precipitates
formed were isolated by vacuum filtration, washed with hot water
1H), 7.97 (t, J=7.36 Hz, 2H), 8.97 (s, 1H) ppm. 13C NMR (100 MHz,
DMSO-d6): δ=20.5, 25.2, 25.3, 32.5, 44.6, 114.7, 122.8, 125.9, 126.1,
128.3, 130.7, 132.6, 132.8, 134.4, 137.0, 147.0, 180.1, 180.2 ppm.
Anal. calcd for C23H23NO2S (377): C, 73.18; H, 6.14; N, 3.71; S, 8.49
Found: C, 73.01; H, 6.48; N, 3.95; S, 8.13.
1
2
3
4
5
°
(500 mL) and dried at 50 C. The resulting solid product was purified
by column chromatography using column silica gel 100–200 mesh
(ethyl acetate:hexane 1:4) to give compounds 5x–5y.
6
7
8
9
Biology
2-chloro-3-(phenylamino)naphthalene-1,4-dione (5x)
In vitro Antibacterial Activity
°
~
Deep maroon solid (96%); mp: 222–223 C; IR (KBr): n=718, 852,
1225, 1298, 1335, 1440, 1549, 1615, 1672, 3250, 3334. 1H NMR
The bacterial strains used including S. aureus (ATCC 6538), E. faecalis
(PCM 2673), E. coli (ATCC 8739), S. bongori (PCM 2552), E. cloacae
(PCM 2569), K. pneumoniae (PCM1), P. aeruginosa (PCM 2562), and
yeast C. albicans (ATCC10231) were collected from the Department
of Molecular Biology, The John Paul II Catholic University of Lublin,
Poland.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
°
(400 MHz, DMSO-d6, 25 C, TMS): δ=7.11-7.14 (m, 3H), 7.31 (t, J=
7.72 Hz, 2H), 7.79 (dt, J=7.56 Hz & J=1.04 Hz, 1H), 7.85 (dt, J=
7.44 Hz & J=1.12 Hz, 1H), 8.02 (d, J=7.60 Hz, 2H), 9.29 (s, 1H) ppm.
13C NMR (100 MHz, DMSO-d6): δ=114.2, 123.8, 124.2, 125.8, 126.2,
127.7, 130.0, 131.8, 132.9, 134.5, 138.6, 142.9, 176.4, 179.8 ppm.
Anal. calcd for C16H10ClNO2 (283): C, 67.74; H, 3.55; N, 4.94 Found: C,
67.42; H, 3.51; N, 4.88; Beilstein test: Cl positive.[36]
The in vitro antimicrobial study was performed using the micro-
broth dilution method against tested organisms, followed by a
previously described method.[37,38] The bacterial culture was inocu-
lated in Mueller Hinton Broth medium (Biocorp, Warsaw, Poland)
2-chloro-3-(p-tolylamino)naphthalene-1,4-dione (5y)
°
and incubated at 37 C and with vigorous shaking (200 rpm) for 24-
°
~
Light maroon solid (98%); mp: 205–206 C; IR (KBr): n=718, 817,
h. Bacterial cell suspensions at initial inoculums of 5×105 in
Mueller-Hinton liquid medium were exposed to the examined
compounds at relevant concentrations (range, 0.001–2 mg/mL) for
1019, 1241, 1287, 1330, 1496, 1517, 1562, 1597, 1676, 3225. 1H NMR
°
(400 MHz, DMSO-d6, 25 C, TMS): δ=2.28 (s, 3H), 7.02 (d, J=8.2 Hz,
2H), 7.11 (t, J=8.2 Hz, 2H), 7.78 (dt, J=7.4 Hz & J=1.1 Hz, 1H), 7.86
(dt, J=7.4 Hz & J=1.3 Hz, 1H), 8.02 (dd, J=7.5 Hz & J=1.4 Hz, 2H),
9.26 (s, 1H) ppm. 13C NMR (100 MHz, DMSO-d6): δ=20.6, 113.2,
124.1, 126.0, 126.5, 128.4, 130.2, 132.0, 133.1, 133.7, 134.8, 136.1,
143.2, 176.6, 180.1 ppm. Anal. calcd for C17H12ClNO2 (297): C, 68.58;
H, 4.06; N, 4.70. Found: C, 68.85; H, 3.97; N, 5.07; Beilstein test: Cl
positive.[36]
°
24-h at 37 C. Simultaneously, the standard antibiotics caspofungin,
amphotericin B, chloramphenicol and streptomycin (as a positive
control) were tested against the bacterial pathogens. The MIC was
defined as the lowest concentration of the compound that
inhibited the visible growth of the microorganism. All the experi-
ments were conducted in triplicate.
The MICs of the tested compounds were determined based on the
broth dilution method as recommended by CLSI, with some minor
modifications.[39] The final concentrations of naphthoquinones and
antibiotics ranged from 2000 μg/mL–0.7 μg/mL. The microtiter
plate wells contained 100 μL of each dilution. Next, 20 μL of yeast
inoculum (OD530-nm =1–5×103) was added to each well, and 100 μL
of uninoculated medium was included as a sterility control (blank).
General Procedure for the Synthesis of Compounds 5z–5aa
Compounds 5x–5y (5 mmol) was dissolved in dry acetone
(300 mL), and cyclohexanethiol (0.611 mL, 5 mmol) was gradually
added to the reaction mixture with constant stirring at room
temperature. Trimethylamine (1 equiv. 0.469 mL) was added and
the mixture was refluxed for 2 h. The resulted mixture was poured
into crushed ice and the solid formed was filtered, washed with hot
°
The plates were incubated at 37 C for 48-h. The MIC was defined as
the lowest concentration of disinfectant that inhibited visible
microorganism growth. All the experiments were performed in
triplicate.
°
water (500 mL), dried at 50 C and purified by column chromatog-
raphy (ethyl acetate:hexane, 1:4) to give the pure compounds 5z–
5aa.
Hemolytic Assay
40 2-(cyclohexylthio)-3-(phenylamino)naphthalene-1,4-dione (5z)
Human blood samples were provided voluntarily by the author of
the manuscript (age 38 years, male). The blood was collected in
sterile tubes containing citrate dextrose solution as an anticoagu-
lant. To separate erythrocytes from plasma, the samples were
41
°
~
Deep blue violet solid (79%); mp: 150–151 C; IR (KBr): n=918,
1258, 1339, 1451, 1668, 3145, 3428. 1H NMR (400 MHz, DMSO-d6,
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
°
25 C, TMS): δ=1.07 (d, J=5.92 Hz, 5H), 1.43–1.54 (m, 5H), 2.95 (s,
°
centrifuged at 500×g for 10 min at 4 C, and the supernatant was
1H), 7.04–7.07 (m, 3H), 7.27 (t, J=7.76 Hz, 2H), 7.77 (dt, J=7.40 Hz
& J=1.04 Hz, 1H), 7.83 (dt, J=7.40 Hz & J=1.12 Hz, 1H), 7.99 (d, J=
7.64 Hz, 2H), 9.02 (s, 1H) ppm. 13C NMR (100 MHz, DMSO-d6): δ=
25.1, 25.2, 32.5, 44.5, 115.8, 122.6, 123.4, 125.9, 126.1, 127.8, 130.7,
132.8, 132.9, 134.4, 139.4, 146.6, 180.1, 180.3 ppm. Anal. calcd for
C22H21NO2S (363): C, 72.70; H, 5.82; N, 3.85; S, 8.82 Found: C, 72.41;
H, 5.98; N, 3.91; S, 8.53.
removed. Next, the erythrocytes were resuspended in PBS buffer
(10 mM phosphate, pH 7.5; 150 mM NaCl) and centrifuged at 500×
°
g for 10 min at 4 C. The washing procedure was repeated until a
transparent supernatant was obtained. After washing, the eryth-
rocytes were resuspended in PBS buffer at a final concentration of
2%. Simultaneously, the compounds were prepared at the MIC
concentration (70 μg/mL) in a final volume of 50 μL DMSO. The
prepared compounds were mixed with 450 μL of the 2%
°
erythrocyte suspension and incubated for 1-h at 37 C. Next, the
2-(cyclohexylthio)-3-(p-tolylamino)naphthalene-1,4-dione (5aa)
samples were centrifuged at 5000×g for 10 min, and absorbance
°
was measured at a wavelength of 415 nm.[1]
~
Black solid (98%); mp: 118–119 C; IR (KBr): n=754, 812, 1108, 1282,
1
°
1458, 1552, 1662, 3142, 3418. H NMR (400 MHz, DMSO-d6, 25 C,
TMS): δ=1.07 (d, J=6.76 Hz, 5H), 1.43–1.54 (m, 5H), 2.26 (s, 3H),
2.95 (s, 1H), 6.96 (d, J=8.24 Hz, 2H), 7.07 (d, J=8.20 Hz, 2H), 7.75
(dt, J=7.52 Hz & J=1.08 Hz, 1H), 7.81 (dt, J=7.40 Hz & J=1.24 Hz,
ChemistryOpen 2019, 8, 589–600
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Full Papers
ROS detection
Acknowledgements
1
2
3
4
5
6
7
8
9
The generation of ROS was investigated using the fluorescent dye
DCFH-DA.[40] S. aureus was cultivated, thoroughly washed with PBS
buffer, and resuspended in PBS buffer (pH 7.2). Bacterial cells were
This work was supported by grants from the Medical Research
Center Program (NRF-2017R1A5A2015061) through the National
Research Foundation (NRF), which is funded by the Korean
government (MSIP). This work was supported by the Korea
Institute of Energy Technology Evaluation and Planning (KETEP)
and the Ministry of Trade, Industry & Energy (MOTIE) of the
Republic of Korea (No. 20184030202210).
°
stained for 30 min with 10 μM DCFH-DA at 37 C under dark
conditions. Next, the cells were washed with water until the
unreacted DCFH-DA was removed. DCFH-DA-treated bacterial cells
were incubated with compounds 5b, 5j, and 5q (70 μg/mL, 70 μg/
mL, and 30 μg/mL, respectively) for 4-h. Lastly, ROS generation in
samples was examined under a fluorescence microscope (LSM 510
META-Carl Zeiss, Jena, Germany) The excitation-emission wave-
lengths were fixed at 488 nm and 535 nm, respectively.[41]
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Conflict of Interest
Bactericidal Time-Kill Kinetic Study
The authors declare no conflict of interest.
The bacteria S. aureus were cultured overnight and diluted to
1:10,000 in MHB medium. The cells were incubated at 37 C and
aerated at 225 rpm for 2 h. The compounds 5b, 5j, and 5q were
treated with bacteria at their respective MIC in culture tubes at
°
Keywords: naphthoquinone derivatives · antimicrobial activity ·
hemolysis · reactive oxygen species · necrosis
°
37 C and 225 rpm. At regular intervals, 100 μL of bacterial solution
was placed in
a 96-well plate, centrifuged for 3 min, and
resuspended in 100 μL of phosphate buffer (1×PBS). Serially diluted
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Manuscript received: February 27, 2019
Revised manuscript received: April 18, 2019
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